Nov 24, 1976 - sional coughing, labored breathing, nasal discharge, and lethargy. Convales- cence was well advanced by the day 10. All monkeys excreted ...
INFICTION AND IMMUNITY, May 1977, p. 476-479 Copyright © 1977 American Society for Microbiology
Vol. 16, No. 2 Printed in U.S.A.
Reaction of Squirrel Monkeys to Intratracheal Inoculation with Influenza/A/New Jersey/76 (Swine) Virus RICHARD F. BERENDT* AND WILLIAM C. HALL U. S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21701
Received for publication 24 November 1976
To determine whether a model could be established for laboratory investigations, nine squirrel monkeys were inoculated intratracheally with 107 median egg-infectious doses of influenza virus type A/New Jersey/8/76 (H1:W1N1) (swine influenza virus). They responded with clinically detectable illness including fever, leukopenia, decreased food consumption, increased respiratory rate, occasional coughing, labored breathing, nasal discharge, and lethargy. Convalescence was well advanced by the day 10. All monkeys excreted virus for 7 to 8 days. A scoring procedure (illness score) has been developed for use in studies of vaccine and chemotherapeutic efficacy.
In February 1976, influenza virus bearing swine antigenic determinants (H,,lNl) was isolated from several recruits at Fort Dix, N.J. (6). Because this virus represented a serotype to which the majority of the American population had no immunity, it was feared that a pandemic might begin during the following fall. Consequently, a decision was made to make vaccine available to all Americans. Although extensive investigations of the antigenicity and adverse reactions after administration of various swine influenza vaccines have already been carried out (5), no reports of the protection afforded by these vaccines have been published. The principal reason for the lack of protection data is that the virulence and communicability of the recently isolated strain is unknown, and, as a consequence, no experimental inoculation of humans with swine influenza has been attempted in the United States. A desire to limit the dissemination of the virus has also largely precluded experiments with laboratory animals. Because we have recently published the details of a squirrel monkey model for influenza infections (3) and because we have unique containment facilities for the study of potentially hazardous microorganisms, we were requested by the National Institute of Allergy and Infectious Diseases to investigate the protective efficacy of vaccines and chemotherapeutic agents in primates. To carry out investigations of prophylaxis, it was first necessary to determine whether the New Jersey strain of influenza virus caused clinical illness in squirrel monkeys and, if so, whether a means of assessing the degree of clinical illness could be developed to compare responses among groups of animals given var476
ious treatments. A systemic reaction score has been used by Parkman et al. to evaluate the reaction of human subjects to various vaccine preparations (5) and by Beare and Craig to evaluate the reaction of humans to various test strains of influenza virus (clinical score) (1). The clinical response (illness score) of squirrel monkeys to intratracheal inoculation of virus is presented in this report. MATERIALS AND METHODS Virus preparation and assay. A sample of the fifth egg passage of influenza A/New Jersey/8176 (HwlNl) was obtained from the Bureau of Biologics, Food and Drug Administration, diluted 1:1,000 in sterile heart infusion broth (HIB), and inoculated into the allantoic cavity at 10- to 12-day embryonated eggs. After 48 h of incubation at 35°C, the allantoic fluid was collected, distributed in 3-ml aliquots, and stored at -70°C. Estimates of the concentrations of virus in terms of egg infectivity were accomplished as previously described (2). The allantoic fluid preparations had a titer of 107.2 median egg-infectious doses per ml. Antigenic purity of the viral preparation was confirmed with reference standard materials supplied by the Center for Disease Control. All serological tests were carried out by standard Center for Disease Control procedures (4). Monkeys. Twenty-one male squirrel monkeys (Saimiri sciureus), obtained from commercial sources, were used in this study. Average weight was 0.7 kg (range, 0.493 to 1.01 kg). Monkeys were housed in wire-bar cages and fed commercial monkey chow supplemented with fruit until the time of the experiment. Water was provided ad libitum. During experiments food was limited to six commercial biscuits daily. Intratracheal inoculation. The procedure for intratracheal inoculation of virus has been described previously (3). Briefly, it involves inoculation
VOL. 16, 1977
SWINE INFLUENZA IN SQUIRREL MONKEYS
through a plastic catheter that has been passed over the epiglottis and into the trachea of a lightly anesthetized monkey. Bacterial and viral isolation. Established methods were used to detect bacteremia (2, 3). Virus was isolated from the oropharynx by swabbing the back of the throat and the tonsil area with a swab moistened with HIB. The contents of the swab were washed into 1.0 ml of HIB containing 50 ,g of gentamicin, 100 U of penicillin, and 100 ,ug of streptomycin per ml. The suspensions were incubated at room temperature for 60 min to kill bacteria and were then inoculated into embryonated eggs (six eggs per sample). After 48 h of incubation at 35°C, followed by overnight refrigeration, the presence of virus was detected by routine hemagglutination procedures. Clinical and laboratory determinations. Rectal temperature, hematocrit, total and differential leukocyte counts, respiratory rate, pharyngeal virus isolation, food and water consumption, body weight, nasal discharge, coughing and sneezing, labored
breathing, and activity were recorded daily. Blood for serum hemagglutination inhibition antibody determinations was obtained before virus inoculation and at 7, 14, and 28 days after infection.
RESULTS Preliminary observations. Nine monkeys were inoculated intratracheally with 107 median egg-infectious doses of virus. Figure 1 presents the deviation from baseline values (baseline values are the mean of three daily observations on each of the nine monkeys) of five of the response parameters. Frequency of occurrence of three more parameters is also illustrated in the same figure. Activity and dyspnea are omitted from the figure because lethargy and labored breathing were seen daily in every monkey. Hematocrit values are not included because of variation due to daily bleeding. Al100 .9
RECTAL TEMPERATURE .-%
*t. o z o
2.0-
80-
1.0
60~ 40_
4 0
0 -
I 1
1
477
RATE
RESPIRATORY
~~~~~~w20-~ O
I I
0
I
v
-I
I
I
4
BODY WEIGHT
0
V
-
60-
-
-5 uj
-
-10 I
FOOD CONSUMPTION
O2040-
at
- 80-
z
4
I 0
LEUKOCYTE CONC.
0
-l0 -20
VIRUS ISOLATION
100
-
50-L2iz _'I_
1001
I
I
COUGH / SNEEZE
50-30 -40
I
10050LJ
I
1
I
NASAL DISCHARGE
Iv
0 1
I
I
I
I
2 3 4 5 6 7
DAYS AFTER INFECTION FIG. 1. Change in selected clinical parameters after intratracheal inoculation of the New Jersey strain of influenza virus. Vertical bars represent the standard error of the mean.
478
INEFECT . IMMUN .
BERENDT AND HALL
TABLE 1. Seven-day illness score for squirrel though sick monkeys seemed to drink less water than healthy ones, it was impossible to mea- monkeys infected with swine influenza virus (A/New Jersey/76) sure water consumption accurately because these monkeys would only drink from bowls Mean score and lost water by splashing. Parameter and scoring procedure for 9 inMonkeys became ill within 24 h after virus keys inoculation; fever was most prominent at this 1. Virus 1 for each that vi8.75 shedding: day (1.25)a time and then slowly subsided. Most of the rus was detected, plus 1 additional for other parameters seemed to reach a maximum each day that -67% of eggs were posi(minimum for leukocyte count) in 2 to 5 days tive 8.0 (1.31) and then slowly subsided. All values had re- 2. Temp: 2 for each day that rectal temperature was -1°F (.0.6aC) above turned to normal within 10 days (not shown); baseline value virus shedding also ceased by this time. At no 3. Body wt: 1 for each percent loss of body 9.9 (1.94) time were bacteria isolated from the blood. At weight at 7 days 10 days the monkeys had also begun to gain 4. Leukocyte concn: 1 for each day that 4.4 (0.84) total leukocyte count was
